Genome reduction boosts heterologous gene expression in Pseudomonas putida

Microbial Cell Factories

Research

Sarah Lieder¹  Ralf Takors¹  Víctor de Lorenzo²  Pablo I Nikel² 

[1] Institute of Biochemical Engineering, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany;Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), C/Darwin 3, 28049, Madrid, Spain;

关键词: Pseudomonas putida;    Reduced genome;    Heterologous gene expression;    Chemostat;    Energy maintenance;    Metabolic engineering;    Microbial cell factory;   

DOI  :  10.1186/s12934-015-0207-7

 received in 2014-11-13, accepted in 2015-02-11,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundThe implementation of novel platform organisms to be used as microbial cell factories in industrial applications is currently the subject of intense research. Ongoing efforts include the adoption of Pseudomonas putida KT2440 variants with a reduced genome as the functional chassis for biotechnological purposes. In these strains, dispensable functions removed include flagellar motility (1.1% of the genome) and a number of open reading frames expected to improve genotypic and phenotypic stability of the cells upon deletion (3.2% of the genome).ResultsIn this study, two previously constructed multiple-deletion P. putida strains were systematically evaluated as microbial cell factories for heterologous protein production and compared to the parental bacterium (strain KT2440) with regards to several industrially-relevant physiological traits. Energetic parameters were quantified at different controlled growth rates in continuous cultivations and both strains had a higher adenosine triphosphate content, increased adenylate energy charges, and diminished maintenance demands than the wild-type strain. Under all the conditions tested the mutants also grew faster, had enhanced biomass yields and showed higher viability, and displayed increased plasmid stability than the parental strain. In addition to small-scale shaken-flask cultivations, the performance of the genome-streamlined strains was evaluated in larger scale bioreactor batch cultivations taking a step towards industrial growth conditions. When the production of the green fluorescent protein (used as a model heterologous protein) was assessed in these cultures, the mutants reached a recombinant protein yield with respect to biomass up to 40% higher than that of P. putida KT2440.ConclusionsThe two streamlined-genome derivatives of P. putida KT2440 outcompeted the parental strain in every industrially-relevant trait assessed, particularly under the working conditions of a bioreactor. Our results demonstrate that these genome-streamlined bacteria are not only robust microbial cell factories on their own, but also a promising foundation for further biotechnological applications.

【 授权许可】

Unknown   
© Lieder et al.; licensee BioMed Central. 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.